Humoral immune response against epidermal growth factor encapsulated in dehydration rehydration vesicles of different phospholipid composition.

We investigated the influence of dehydration-rehydration vesicles (DRV) phospholipid composition and the addition of other components on human recombinant epidermal growth factor (hrEGF) encapsulation efficiency and its release from liposomes. Encapsulation of EGF into DRV composed of phosphatidylcholine with different unsaturation levels was around 20-35%. The best result was obtained with dipalmitoyl phosphatidylcholine: cholesterol (DPPC:Ch) liposomes (35%) corresponding to the lowest hrEGF release during one month of storage. Even with this phospholipid composition, modification of the DRV procedure by including an extrusion step did not improve hrEGF encapsulation efficiency, rendering less stable particles. The inclusion of recombinant P64k from Neisseria meningitidis (rP64k), as such or conjugated to hrEGF, decreased the encapsulation efficiency of the latter protein into DRV or freeze and thaw multilamellar vesicles (FATMLV). The hrEGF release from liposomes could be related to the interaction between this polypeptide and the bilayer, as evidenced by increased carboxyfluorescein release from hrEGF-DRV; less susceptibility to fluorescence quenching by acrylamide in the presence of liposomes; and a measurable decrease of phospholipid phase transition Delta enthalpy (DeltaH). DRV comprising saturated phospholipids (DPPC:Ch or distearoyl phosphatidylcholine [DSPC]:Ch) and containing the conjugate EGF-P64k induced a more efficient immune response against hrEGF than unsaturated phospholipid and alum in terms of total IgG, IgG(2a), and IgG(2b) subclasses and the ability of antibody to inhibit the interaction of the EGF receptor with hrEGF.

Liposome-mediated DNA vaccination: the effect of vesicle composition.

Liposome-entrapped DNA has been shown to enhance the potency of DNA vaccines, possibly by facilitating uptake of the plasmid by antigen-presenting cells (APC). In this paper, we have investigated the influence of the liposomal composition and surface charge on such potency. Plasmid DNA pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen was entrapped within cationic liposomes of various compositions and surface charges with high efficiency (88-97% of the amount used) by the dehydration-rehydration method that generates dehydration-rehydration vesicles (DRV). Cryo-electron microscopy revealed that DNA-containing DRV (DRV(DNA)) were multilamellar. In immunisation studies, female Balb/c mice were given two to four intramuscular injections of 10 microg naked or liposome-entrapped pRc/CMV HBS and bled at time intervals. Results indicate that the lipid composition of the DRV(DNA) influences the strength of the humoural response (immunoglobulin (Ig)G subclasses) with inclusion of dioleoyl phosphatidylethanolamine (DOPE) or phosphatidylethanolamine (PE) in the liposomal structure contributing to greater responses. DRV(DNA) in which the DOPE or PE were omitted or substituted with cholesterol led to significant reduction of humoural responses against the encoded antigen. Replacing phosphatidylcholine (PC) in the DRV(DNA) with the high-melting distearoyl phosphatidylcholine also contributed to lower responses. In other experiments, IgG responses were monitored in mice immunised with pRc/CMV HBS entrapped in DRV composed of PC and DOPE as before but incorporating increasing amounts of DOTAP (1-16 micromol). Maximal IgG responses were observed at 10 weeks after the first of four injections and suggested a trend of higher responses when 4 or 8 micromol DOTAP was present in the DRV(DNA) formulation. Cell-mediated immunity (measured in terms of endogenous antigen-specific splenic interferon-gamma) in mice immunised with pRc/CMV HBS entrapped in liposomes composed of PC, DOPE and DOTAP (16:8:4 molar ratio) was much greater than in animals treated with naked plasmid. These results indicate that liposome-mediated DNA immunisation is more effective than the use of naked DNA, and also suggest that the presence of fusogenic phosphatidylethanolamine in DRV in conjunction with a low-melting phosphatidylcholine and an appropriate content of cationic lipid might contribute to more effective liposomal DNA vaccines. The notion that liposomes improve immune responses to the plasmid-encoded vaccine by facilitating the latter's uptake by APC was supported by the observation that in Balb/c mice injected intramuscularly with liposome-entrapped pCMV. Enhanced green fluorescent protein, expression of the gene in terms of fluorescence intensity in the draining lymph nodes, was much greater than in animals treated with the naked plasmid.

The effect of polysialylation on the immunogenicity and antigenicity of asparaginase: implication in its pharmacokinetics.

Erwinia carotovora L-asparaginase was conjugated via the epsilon-amino groups of its lysine residues with colominic acid (CA) (polysialic acid) of average molecular mass of 10 kDa by reductive amination in the presence of NaCNBH3. Polysialylation using 50-, 100- and 250-fold molar excess CA relative to the enzyme led to an increasing proportion of the enzyme's in-amino groups (5.8, 7.6 and 11.3%, respectively) being conjugated to CA. Polysialylated and native (intact) asparaginase were used to immunize mice intravenously. Results (total IgG immune responses) indicate that all preparations elicited antibody production against the enzyme moiety but not against the CA of the conjugates. Moreover, antibody titres appeared highest for the native enzyme and were generally reduced as the degree of polysialylation increased. In other experiments mice pre-immunized with native or polysialylated asparaginase, with anti-asparaginase antibodies in their blood, were injected intravenously with the corresponding enzyme preparations. Results revealed that polysialylation reduces the antigenicity of asparaginase thus leading to circulatory half-lives (t 1/2 beta) that were 3-4-fold greater than that of the native enzyme, and similar to those observed in naive, non-immunized mice. Our data suggest that polysialylation of therapeutic enzymes and other proteins may be useful in maintaining their pharmacokinetics in individuals with antibodies to the therapeutic proteins as a result of chronic treatment.

Liposome-entrapped plasmid DNA: characterisation studies.

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.

Immunogenicity of plasmid DNA encoding the 62 kDa fragment of Schistosoma japonicum myosin.

The recombinant Schistosoma mansoni 62 kDa myosin fragment, rIrV-5, is highly protective in experimental animals, however, vaccination of mice and rats with the recombinant Schistosoma japonicum homologue, rSj62, did not induce significant resistance against S. japonicum infection. To explore alternative ways of presenting this antigen, we further constructed a plasmid (VRSj62) which encodes Sj62 using the VR1020 vector and tested it in vaccination experiments. Four immunisations with 10 microg VRSj62 DNA alone were sufficient to induce high and progressively increasing levels of IgG antibodies against rSj62 with increasing numbers of injections in CBA/Ca mice (IgG titre > or =1:25000), and three injections with 50 microg VRSj62 DNA alone induced significant IgG responses in C57Bl/6 mice (IgG titre, 1:1600). However, vaccination with plasmid DNA entrapped in cationic liposomes or together with pUC19 DNA as a source of CpG motifs, both of which have been reported to enhance immune responses, did not enhance specific antibody production. In spite of the stimulation of specific antibodies against rSj62 with the naked DNA construct no resistance to challenge was demonstrated.

Polysialic acids: potential in improving the stability and pharmacokinetics of proteins and other therapeutics.

Naturally occurring polymers of N-acetylneuraminic acid (polysialic acids) are biodegradable, highly hydrophilic and have no known receptors in the body. Following intravenous injection, polysialic acids exhibit long half-lives in the blood circulation and have therefore been proposed as carriers of short-lived drugs and small peptides. In addition, shorter-chain polysialic acids can be used as a means to increase the circulatory half-life of proteins and thus serve as an alternative to the nonbiodegradable monomethoxypoly(ethylene glycol). Recent work has shown that covalent coupling of a low molecular weight polysialic acid (colominic acid) to catalase and asparaginase leads to a considerable increase of enzyme stability in the presence of proteolytic enzymes or blood plasma. Comparative studies in vivo with polysialylated and intact asparaginase revealed that polysialylation significantly increases the half-life of the enzyme. The highly hydrophilic and innocuous nature of polysialic acids renders them suitable as a means to prolong the circulation of peptides and proteins.

Entrapment of plasmid DNA vaccines into liposomes by dehydration/rehydration.

Intramuscular injection of naked plasmid DNA is known (1-3) to elicit humoral and cell-mediated immune responses against the encoded antigen. It is thought (2,3) that immunity follows DNA uptake by muscle cells, leading to the expression and extracellular release of the antigen which is then taken up by antigen presenting cells (APC). In addition, it is feasible that some of the injected DNA is taken up directly by APC. Disadvantages (1-3) of naked DNA vaccination include: uptake of DNA by only a minor fraction of muscle cells, exposure of DNA to deoxyribonuclease in the interstitial fluid thus necessitating the use of relatively large quantities of DNA, and, in some cases, injection into regenerating muscle in order to enhance immunity. We have recently proposed (1,4) that DNA immunization via liposomes (phospholipid vesicles) could circumvent the need of muscle involvement and instead facilitate (5) uptake of DNA by APC infiltrating the site of injection or in the lymphatics, at the same time protecting DNA from nuclease attack (6). Moreover, transfection of APC with liposomal DNA could be promoted by the judicial choice of vesicle surface charge, size and lipid composition, or by the co-entrapment, together with DNA, of plasmids expressing appropriate cytokines (e.g., interleukin 2), or immunostimulatory sequences.

Vaccine entrapment in liposomes.

The use of liposomes as carriers of peptide, protein, and DNA vaccines requires simple, easy-to-scale-up technology capable of high-yield vaccine entrapment. Work from this laboratory has led to the development of techniques that can generate liposomes of various sizes, containing soluble antigens such as proteins and particulate antigens (e.g., killed or attenuated bacteria or viruses), as well as antigen-encoding DNA vaccines. Entrapment of vaccines is carried out by the dehydration-rehydration procedure which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free vaccines. On rehydration, the large multilamellar vesicles formed incorporate up to 90% or more of the vaccine used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped vaccine still associated with the vesicles. A similar technique applied for the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4-5 microm in diameter) in the presence of spores. On rehydration and sucrose gradient fractionation of the suspension, up to 30% or more of the spores used are associated with generated giant liposomes of similar mean size.

Sorbitan monostearate/polysorbate 20 organogels containing niosomes: a delivery vehicle for antigens?

Multi-component organogels formed using the non-ionic surfactant sorbitan monostearate as gelator have been formulated to contain niosomes. The purpose of this study was to evaluate the potential of these vesicle-in-water-in-oil (v/w/o) gels as delivery vehicles for vaccines. Bovine serum albumin (BSA) and haemagglutinin (HA) were used as model antigens in depot and immunogenicity studies respectively. The complex gels were prepared by the addition of a hot (60 degrees C) aqueous niosome suspension (v/w) to the sol phase (o, an organic solution of the gelator); a vesicle-in-water-in-oil (v/w/o) emulsion was produced which cools to an opaque, semi-solid, thermoreversible v/w/o gel. Light microscopy of the organogel revealed that the microstructure consists of a tubular network of surfactant aggregates in the organic medium, the niosome suspension being dispersed in these surfactant tubules. Therefore, in such gels, the vaccine is thought to be entrapped in the niosomes, themselves located within the sorbitan monostearate tubular network in the organic medium. In vivo, a depot effect was observed following intramuscular administration of the gel containing the entrapped bovine serum albumin, cleared from the injection site over a period of days. The relatively short-lived nature of the depot was thought to arise due to interactions between the gel and the local interstitial fluid which results in gel disintegration in situ. Thus, the niosomes containing antigens are believed to be released from the organic gel. Immunogenicity studies showed that the v/w/o gel as well as one of the controls, the water-in-oil (w/o) gel, possess immunoadjuvant properties and enhance the primary and secondary antibody titres (of total IgG, IgG1, IgG2a and IgG2b) to haemagglutinin antigen. As far as humoral immunity is concerned, the w/o gel showed stronger immunoadjuvant properties compared to the v/w/o gel, being effective at a lower antigen dose i.e 0.1 microg HA.

Interaction of a nonionic surfactant-based organogel with aqueous media.

In an attempt to explain the rather short half-life of molecules at the injection site after their intra-muscular administration in a sorbitan monostearate organogel, in vitro studies were carried out to study the effects of an aqueous medium (simulating interstitial fluid at injection site) on the physical form of the organogel. When the gel mass comes in contact with an aqueous phase, the latter penetrates into the organic gel via the sorbitan monostearate tubular network, resulting in gel breakdown into smaller fragments. The surfactant tubular network act as a conduit for water penetration into the gel. Meanwhile, emulsification, aided by the surfactants present in the gel, also occurs at the gel surface between the organogel and the aqueous phase. This leads to a gradual erosion of the gel as oil droplets bud off from the gel mass. From these in vitro observations, we speculate that after gel administration in vivo, dynamic interactions occur between the local interstitial fluid and the gel mass: fluid penetration into the gel and emulsification at the gel surface is thus responsible for gel breakdown and so a relatively short duration of drug at the injection site.

Novel sorbitan monostearate organogels.

Sorbitan monostearate, a hydrophobic nonionic surfactant, gels a number of organic solvents such as hexadecane, isopropyl myristate, and a range of vegetable oils. Gelation is achieved by dissolving/dispersing the organogelator in hot solvent to produce an organic solution/dispersion, which, on cooling sets to the gel state. Cooling the solution/dispersion causes a decrease in the solvent-gelator affinities, such that at the gelation temperature, the surfactant molecules self-assemble into toroidal inverse vesicles. Further cooling results in the conversion of the toroids into rod-shaped tubules. Once formed, the tubules associate with others, and a three-dimensional network is formed which immobilizes the solvent. An organogel is thus formed. Sorbitan monostearate gels are opaque, thermoreversible semisolids, and they are stable at room temperature for weeks. The gels are affected by the presence of additives such as the hydrophilic surfactant, polysorbate 20, which improves gel stability and alters the gel microstructure from a network of individual tubules to star-shaped "clusters" of tubules in the liquid continuous phase. Another solid monoester in the sorbitan ester family, sorbitan monopalmitate, also gels organic solvents to give opaque, thermoreversible semisolids. Like sorbitan monostearate gels, the microstructure of the palmitate gels comprise an interconnected network of rodlike tubules. Unlike the stearate gels, however, the addition of small amounts of a polysorbate monoester causes a large increase in tubular length instead of the "clustering effect" seen in stearate gels. The sorbitan stearate and palmitate organogels may have potential applications as delivery vehicles for drugs and antigens.

Water-in-sorbitan monostearate organogels (water-in-oil gels).

Novel multicomponent organogels containing an aqueous phase are described, and some properties which influence their potential as delivery devices for hydrophilic drugs and vaccines are discussed. The gel is produced by preparing a hot water-in-oil (w/o) emulsion using sorbitan monostearate, a nonionic surfactant which is also the organogelator, as the principal emulsifying agent. On cooling at room temperature, the w/o emulsion sets to an opaque, semisolid, thermoreversible organic gel. Cooling the emulsion results in a reduced solubility of the sorbitan monostearate in the oil, with a corresponding decrease in solvent-surfactant affinities, causing surfactant self-assembly into aggregates. The microstructure of the w/o gel is seen by light microscopy to consist of a network of tubules and fibrils (containing the aqueous phase) dispersed in the organic medium. X-ray diffraction and freeze-fracture studies suggest that the tubular aggregates in the w/o gel are made up of surfactant molecules arranged in inverted bilayers and that the aqueous phase is accommodated within these inverted bilayers, bound by the polar headgroups of the surfactant molecules. The presence of water in the tubular skeleton of the organic gels results in the establishment of percolating electroconductive aqueous channels in the organogel. Increasing the water content of a w/o gel causes the surfactant tubules to swell with a corresponding increase in conductivity until the tubules are saturated. Further increase in the water content results in the excess water accumulating in droplets within the organic medium and a decrease in conductivity as the gel integrity is compromised. The w/o gels (containing a model antigen, radiolabeled bovine serum albumin, in the aqueous phase) have demonstrated depot properties after intramuscular administration to mice, entrapped antigen being released over a period of days.

Inverse toroidal vesicles: precursors of tubules in sorbitan monostearate organogels.

Sorbitan monostearate organogels are opaque, thermoreversible semi-solids whose microstructure consists of surfactant tubules dispersed in the organic continuous phase. Inverse toroidal vesicles are the precursors of the surfactant tubules. The gelation process was observed as an isotropic sol phase of sorbitan monostearate in isopropyl myristate was cooled using hot-stage light microscopy. At the gelation temperature, inverse toroidal vesicular structures were seen to grow in the organic phase. These toroids are thought to be analogous to other well-known vesicles, liposomes and niosomes, except for their toroidal (rather than spherical) shape and their inverse nature. They are rather short-lived structures: on further cooling of the sol phase, tubules form in the organic medium: it is speculated that the toroids elongate into tubular shapes or split into rod-shaped segments.

DNA vaccines: a role for liposomes.

Vaccination with attenuated or killed microbes, purified or recombinant subunit proteins and synthetic peptides is often hampered by toxicity, the presence of infectious agents, weak immune responses and prohibiting costs, especially in the developing world. Such problems may be circumvented by genetic immunization, which, by the use of plasmid DNA encoding antigens from bacteria, viruses, protozoa and cancers leads to protective humoral and cell-mediated immunity. This review deals with the background and progress made so far with DNA vaccines and evaluates the role of liposomes in their optimization.

Biodistribution, stability, and antiviral efficacy of liposome-entrapped phosphorothioate antisense oligodeoxynucleotides in ducks for the treatment of chronic duck hepatitis B virus infection.

This study investigated the feasibility of using liposomes to increase the hepatic delivery and antiviral efficacy of phosphorothioate antisense oligodeoxynucleotides (PS-ODN) for the in vivo treatment of hepatitis B virus (HBV) infection. Ducks infected with duck hepatitis B virus (DHBV) were used as the model. We studied the stability of an antisense PS-ODN in duck plasma, its integrity during the process of liposome entrapment, its in vivo biodistribution, plasma clearance, and excretion. In addition, the intrahepatic distribution of a labeled free and liposome-entrapped ODN was also investigated. The results of our studies show that: 1) phosphorothioate ODN remain stable during the process of liposome entrapment; 2) are stable in duck plasma for many hours; 3) are rapidly cleared from the plasma when injected intravenously; 4) intravenous injection of antisense ODNs entrapped within liposomes enhances delivery of the ODN to the liver; and 5) inhibit DHBV replication. Serum DHBV DNA levels fell rapidly, with a corresponding decrease in intrahepatic viral replicative intermediates at the end of the 5-day study period. Although inhibition of viral replication and a fall in the target protein was observed, a marked inhibition of viral replication was also observed with high doses of a random-sequence ODN. Thus, it is not certain that inhibition of viral replication was entirely through an antisense mechanism. Therefore, liposomes may be effective vehicles to improve the delivery of antisense oligonucleotides to the liver for the therapy of hepatotropic viruses.

Genetic vaccines: strategies for optimization.

Vaccination with attenuated or killed microbes, purified or recombinant subunit proteins and synthetic peptides is often hampered by toxicity, the presence of infectious agents, weak immune responses and prohibiting costs, especially in the developing world. Such problems may be circumvented by genetic immunization which has recently emerged as an attractive alternative to conventional vaccines. Numerous studies have already shown that immunization of experimental animals with plasmid DNA encoding antigens from a wide spectrum of bacteria, viruses, protozoa and cancers leads to protective humoral and cell-mediated immunity. This review deals with the background and progress made so far with DNA vaccines and their theoretical and practical advantages as well as potential risks, discusses proposed mechanisms of DNA transfection of cells and induction of immune responses to the produced vaccine antigen, and evaluates strategies for the control and optimization of such responses.

Morphological observations on liposomes bearing covalently bound protein: studies with freeze-fracture and cryo electron microscopy and small angle X-ray scattering techniques.

The appearance of protein bound to the surface of intact and microfluidized liposomes and its possible influence on their morphology was examined by freeze-fracture electron microscopy, cryo electron microscopy and small angle X-ray scattering (SAXS) techniques. Results obtained by the two microscopy techniques were in agreement with one another in terms of vesicle size and localization of protein (tetanus toxoid or immunoglobulin G) on the surface of vesicles. Surface-bound protein was observed as particles (10-12 nm diameter) by freeze-fracture electron microscopy and was confirmed by immunogold cryo microscopy. SAXS was shown to be a suitable means to further characterize liposomes with, or without bound protein.

The immunological co-adjuvant action of liposomal interleukin-2: the role of mode of localisation of the cytokine and antigen in the vesicles.

In experiments designed to study the co-adjuvant action of interleukin-2, a model antigen (tetanus toxoid) was passively entrapped in, or covalently coupled to multilamellar liposomes in the presence or absence of interleukin-2 (IL-2). When present, IL-2 was either co-entrapped with the toxoid, entrapped alone in liposomes with toxoid coupled to their surface, or coupled to the surface of liposomes with entrapped toxoid. The role of spatial localization of IL-2 within the liposomal structure (vis a vis that of the toxoid) was studied in terms of its immunoadjuvant action in vivo. Male CD-1 mice were injected intramuscularly twice with a variety of toxoid-containing liposomal preparations in the absence or presence of IL-2 incorporated in the same liposomes as above. In some experiments mice were immunized with liposomal toxoid mixed with separately entrapped IL-2. Results show that IL-2 augments significantly secondary immune responses (IgG1, IgGa, IgG2b subclasses) against the liposomal toxoid (up to 15-fold compared with the liposomal toxoid alone), regardless of cytokine and antigen mode of accommodation in the liposomal structure but only when both are present in the same vesicles. It is suggested that liposomal IL-2 may prove useful as a co-adjuvant for vaccines which are weak or ineffective.